issue during refolding
Hi,
I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein shows
good activity after refolding but the major concern is the 'precipitation'.
Though the protein express
Thank you all for your valuable suggestions. I will try to improve the
process accordingly.
Best,
Dipankar
On Thu, Sep 21, 2017 at 3:18 PM, Tom Murray-Rust
wrote:
> Hi Dipankar,
>
> I've produced several serine protease constructs in Ecoli and then
> refolded them. I would investigate the foll
Hi Dipankar,
I've produced several serine protease constructs in Ecoli and then refolded
them. I would investigate the following (some of which echoes previous
comments):
- try both urea and guanidine as your denaturant
- try refolding in stages (e.g. 8M urea to 2M to 100mM or equivalent)
- defin
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, September 21, 2017 3:42:28 PM
Subject: [ccp4bb] Precipitation issue during refolding
Hi,
I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein shows
good acti
Hi Dipankar -
I will echo Thuy's suggestion of higher glycerol concentration in your
refolding buffer. A protocol I used in a previous lab (for refolding antibody
ScFv fragments) steps down gradually over 3 days from 10%-5%-0% glycerol in the
dialysis buffer, from an initial sample of inclusi
Gesendet: Donnerstag, 21. September 2017 12:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Precipitation issue during refolding
Hi,
I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein shows
good activity
Dear Manna,
I can see that you use only 5% glycerol in your buffer. This concentration is
quite low for non-stable protein. I would suggest you to increase glycerol up
to 20%. I had a case before that I need to use 30% glycerol in order to
decrease the precipitation. Also, how did you do the re
Hey Dipankar,
There are various additives for improving solubility. I would consider some of
these:
- denaturants (ie urea, guanidine)
- aggregation suppressors (arginine, low concentrations of urea, etc)
- folding enhancers (sucrose, ammonium sulfate)
And have you tried refolding your protein
Hi,
I am working with a serine protease. As the protein is not soluble I am
purifying it from the inclusion bodies followed by refolding. The protein
shows good activity after refolding but the major concern is the
'precipitation'. Though the protein express quite well, but I loose almost
50-60% p
